Biosensor

ABSTRACT

The present invention provides a biosensor that enables highly-accurate measurement of a sample solution including a solid component like hemocytes and has a little variation in response. The biosensor includes: an insulating base plate, an electrode system having at least a working electrode and a counter electrode provided on the base plate, a cover member that is combined with the base plate to define a sample solution supply pathway for leading a sample solution from a sample supply unit to the electrode system, a reaction reagent system including at least an oxidation-reduction enzyme and an electron mediator, and a filter disposed between the electrode system and the sample supply unit in the sample solution supply pathway. The biosensor has a space that encircles surface of the filter in an area from one end of the filter close to the sample supply unit to the other end of the filter close to the electrode system. This arrangement effectively prevents the solid component like hemocytes from flowing into the electrode system without being filtered out by the filter.

TECHNICAL FIELD

[0001] The present invention relates to a biosensor that carries outhigh-speed, highly-accurate, simple determination of a target object ina sample.

BACKGROUND ART

[0002] A biosensor has been proposed to determine a specific componentin a sample by a simple procedure without any dilution or stirring thesample solution (Japanese Laid-Open Patent Publication No. 2-062952).

[0003] In this biosensor, an electrode system including a measurementelectrode or a working electrode, a counter electrode, and a referenceelectrode is formed on an insulating base plate, for example, by screenprinting. An enzyme reaction layer including a hydrophilic polymer, anoxidation-reduction enzyme, and an electron mediator is then formed onthe electrode system. A buffer may be added to this enzyme reactionlayer according to the requirements.

[0004] When a sample solution containing a substrate is added dropwiseonto the enzyme reaction layer in the biosensor thus constructed, theenzyme reaction layer is dissolved to cause a reaction of the enzymewith the substrate, which results in reduction of the electron mediator.After completion of the enzyme reaction, the reduced electron mediatoris oxidized electrochemically, and the concentration of the substrateincluded in the sample solution is calculated from the observed value ofoxidation current.

[0005] In principle, the biosensor is applicable to measurement ofdiverse substances by selecting an appropriate enzyme that reacts with atarget substance of measurement as the substrate. For example, whenglucose oxidase is used as the oxidation-reduction enzyme, the biosensoris constructed to measure the concentration of glucose in blood. This iswidely used as a glucose sensor. Application of cholesterol oxidasegives a biosensor that measures cholesterol in serum.

[0006] The value of serum cholesterol generally used for the index ofdiagnosis is the sum of the concentrations of cholesterol andcholesterol ester. The cholesterol ester is, however, not the substrateof the oxidation reaction with cholesterol oxidase. In order to measurethe value of serum cholesterol as the index of diagnosis, an additionalprocess is thus required to change the cholesterol ester to cholesterol.Cholesterol esterase is used as the enzyme that catalyzes this process.

[0007] The biosensor including cholesterol esterase and cholesteroloxidase in its enzyme reaction layer is used to measure the totalconcentration of cholesterol in serum.

[0008] The measurement of cholesterol is affected by cholesterol that ispresent in the cell membrane. The coexistence of a surface active agentwith cholesterol esterase in the reaction reagent layer is preferable toenhance the reactivity. The surface active agent destroys the cellmembrane in many cases, and there is a possibility that the substancesinside the cell directly or indirectly affect the enzyme reaction or theelectrode reaction. From this point of view, it is preferable that theenzyme reaction and the subsequent electrode reaction proceed in plasmaor serum in the cholesterol sensor. In biosensors other than thecholesterol sensor, the presence of hemocytes in blood may also affectthe response. It is accordingly ideal that the enzyme reaction and theelectrode reaction proceed in a solution free of hemocytes.

[0009] Centrifugation is a known method to separate plasma or serum fromwhole blood. The centrifugation method, however, takes a rather longtime and requires complicated operations.

[0010] U.S. Pat. No. 3,607,092 discloses a membrane used for testingblood. This membrane has a thin film layer that has permeability toliquids but impermeability to solids like hemocytes and giant moleculeslike protein. Namely this thin film functions to filter out thehemocytes. However, since the solid component is accumulated on the thinfilm with the passage of blood, a large area of the thin film layer isrequired to obtain filtrate of a certain amount sufficient for thereaction of the biosensor. The above-mentioned thin film is thus notsufficient.

[0011] U.S. Pat. No. 4,477,575 discloses an apparatus for and a methodof separating serum from whole blood passing through a glass fiberfilter. The method of separating serum from whole blood with a fiber orporous filter is applicable to the biosensor. This method, however, doesnot make the hemocytes kept in the filter but simply slows down the flowof hemocytes for separation of plasma. In the case of application ofthis method to the biosensor, a certain quantity of filtered plasma orserum sufficient for the reaction in the biosensor should be obtained,before the hemocytes are flown out of the filter. For this purpose, aspecific setting that satisfies this condition should be applied for thelength of the filter in the direction of blood flow.

[0012] The filter satisfying this condition is disposed between oneportion of the biosensor with the electrode system and the reactionreagent system and another portion of the biosensor for supplying bloodas a sample to construct the biosensor having the ability of filteringthe hemocytes. FIG. 9 shows a biosensor of such construction. FIG. 9 isa decomposed perspective view of the biosensor without the reactionreagent layer.

[0013] In the example of FIG. 9, silver paste is printed on aninsulating base plate 101 composed of polyethylene terephthalate byscreen printing to form leads 102 and 103 and the base of an electrodesystem. Conductive carbon paste including a resin binder is printed onthe base plate 101 to form the electrode system including a workingelectrode 104 and a counter electrode 105, while insulting paste isprinted to form an insulating layer 106. The working electrode 104 isconnected to the lead 102, and the counter electrode 105 to the lead103. The insulating layer 106 makes the exposed area of the workingelectrode 104 and the counter electrode 105 constant, and partly coversthe leads.

[0014] The process arranges the insulating base plate 101 with theelectrode system, a cover 108 with an air vent 109, a spacer 107, and afilter 111 having the ability of filtering hemocytes at the positionalrelationship shown by the one-dot chain line and joins together toassemble a biosensor. The filter 111 is cut to fit a sample solutionsupply pathway, which is defined by a slit 110 of the spacer 107 betweenthe cover 108 and the insulating base plate 101. Numeral 113 arepresents a portion at which the filter 111 is in contact with theinsulating base plate. The filter 111 is disposed between the electrodesystem and a sample supply unit 112 on the base plate without coveringover the electrode system including the working electrode 104 and thecounter electrode 105 in the sample solution supply pathway.

[0015] In the biosensor having the above construction, blood addeddropwise onto the sample supply unit 112 soaks into an end of the filter111 close to the sample supply unit. In the filter, the permeation rateof hemocytes is less than the permeation rate of plasma as the liquidcomponent, and the plasma accordingly soaks out of the end of the filterclose to the electrode system. The soak-out plasma dissolves reactionreagents, which include enzymes and are carried at a specific positioncovering over the electrode system or on the rear face of the coverimmediately above the specific position, and fills the whole samplesolution supply pathway from the vicinity of the electrode system to theair vent 109. When the whole sample solution supply pathway is filledwith the liquid, the flow of the liquid in the filter 111 stops, so thatthe hemocytes do not reach the end of the filter close to the electrodesystem but are retained at the current position.

[0016] Through the filtration of hemocytes, the reaction reagent layerdissolved in plasma chemically reacts with a target component includedin the plasma, cholesterol in the case of the cholesterol sensor. Afterelapse of a preset time, the value of electric current is measured bymeans of the electrode reaction. This determines the component in theplasma.

[0017] In this prior art biosensor, part of the blood added dropwise tothe sample supply unit 112 is not absorbed through the end of the filter111 close to the sample supply unit. But the blood including hemocytesis transferred through the gap between the sample solution supplypathway and the filter 111 to reach the reaction reagent layer. Thiscauses the hemocytes or some component in the hemocytes to react withthe reaction reagent and give a significant error to the measurement.

[0018] Bonding the filter 111 to the sample solution supply pathway viaan adhesive may prevent the transfer of blood through the gap betweenthe filter 111 and the sample solution supply pathway.

[0019] The adhesive may, however, affect the blood components. Thismethod also requires application of the adhesive on either the surfaceof the filter 111 or the sample solution supply pathway, which resultsin the complicated manufacturing process.

[0020] The object of the present invention is thus to solve thedrawbacks discussed above by improving a biosensor with a filter that iscapable of filtering a solid component like hemocytes.

[0021] More specifically the object of the present invention is toprovide a biosensor that has stable response by allowing a sample addedto the sensor to soak into a filter and making only a sample solutiontransmitted through the filter reach a reaction reagent layer and anelectrode system.

DISCLOSURE OF INVENTION

[0022] A biosensor in accordance with the present invention includes: aninsulating base plate, an electrode system that is provided on the baseplate and has at least a working electrode and a counter electrode, acover member that is combined with the base plate to define a samplesolution supply pathway for leading a sample solution from a samplesupply unit to the electrode system, a reaction reagent system includingat least an oxidation-reduction enzyme and an electron mediator, and afilter disposed between the electrode system and the sample supply unitin the sample solution supply pathway, the biosensor having a space thatencircles surface of the filter in an area from one end of the filterclose to the sample supply unit to the other end of the filter close tothe electrode system.

[0023] In one preferred mode of the present invention, the sample supplyunit is provided on the base plate, and the sample solution supplypathway is formed along the base plate and the cover member.

[0024] In this mode, it is desirable that the space surrounding thesurface of the filter has a width of not less than 0.5 mm. The width ofthe space less than 0.5 mm may cause blood transmitted through a gapbetween the base plate and/or the cover member forming the samplesolution supply pathway and the filter to reach the area of the space bymeans of capillarity. The preferable width of the space ranges from 0.5mm to 5.0 mm. The width over 5.0 mm may undesirably lead to deformationof the filter under vibrations applied to the sensor. More specifically,the preferable width is 1.0 mm to 3.0 mm.

[0025] In another preferred mode of the present invention, the samplesupply unit is provided on the cover member, and the sample solutionsupply pathway is disposed in a direction of gravity from the samplesupply unit. In this mode, it is preferable that the width of the spacesurrounding the surface of the filter is not less than 100 μm and issmaller than the thickness of the filter.

[0026] The filter used here is a porous body having spaces connectingwith one another in a three-dimensional manner, and the porous bodymoves blood from the sample supply unit toward the sample solutionsupply pathway by capillarity while functions to filter hemocytes basedon a difference between flow resistances of plasma and the hemocytes. Anon-woven fabric preferably composed of a hydrophilic fiber, such asfiber glass, cellulose, or pulp, filter paper, or another porous bodymay be applied for the filter.

[0027] The arrangement of the present invention is preferably appliedfor a cholesterol sensor in which the oxidation-reduction enzyme ischolesterol oxidase.

[0028] In the cholesterol sensor, it is preferable that the reactionreagent system includes an enzyme having an ability of hydrolyzingcholesterol ester. It is also preferable that the enzyme having theability of hydrolyzing cholesterol ester is cholesterol esterase andthat the reaction reagent system includes a surface active agent.

[0029] It is desirable that part or all of the cover member and theinsulating base plate are transparent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a decomposed perspective view illustrating a biosensorwithout a reaction reagent layer in one embodiment of the presentinvention.

[0031]FIG. 2 is a vertical sectional view illustrating the biosensor ofFIG. 1.

[0032]FIG. 3 is a plan view illustrating a main part of a biosensor inanother embodiment of the present invention.

[0033]FIG. 4 is a vertical sectional view illustrating a biosensor instill another embodiment of the present invention.

[0034]FIG. 5 is a vertical sectional view illustrating a biosensor inanother embodiment of the present invention.

[0035]FIG. 6 is a decomposed perspective view illustrating the biosensorof FIG. 5.

[0036]FIG. 7 is a vertical sectional view illustrating a biosensor instill another embodiment of the present invention.

[0037]FIG. 8 is a decomposed perspective view illustrating the biosensorof FIG. 7.

[0038]FIG. 9 is a decomposed perspective view illustrating a prior artbiosensor without a reaction reagent layer.

BEST MODES FOR CARRYING OUT THE INVENTION

[0039] As discussed above, a biosensor of the present invention has asample solution supply pathway that is defined by a combination of abase plate and a cover member, and a filter that is disposed in thesample solution supply pathway between a sample supply unit provided oneither the base plate or the cover member and an electrode system on thebase plate, the biosensor having a space surrounding the surface of thefilter in an area from one end of the filter close to the sample supplyunit to the other end of the filter close to the electrode system.Namely there is a specific area, in which the whole circumference of thesurface of the filter is not in contact with either of the base plateand the cover member that form the sample solution supply pathway.

[0040] One aspect of the present invention is a biosensor, whichincludes: an insulating base plate, an electrode system that is providedon the base plate and has at least a working electrode and a counterelectrode, a cover member that is combined with the base plate to definea sample solution supply pathway for leading a sample solution from asample supply unit on the base plate to the electrode system on the baseplate, a reaction reagent system that includes at least anoxidation-reduction enzyme and an electron mediator and is provided onor in the vicinity of the electrode system, and a filter disposedbetween the electrode system and the sample supply unit in the samplesolution supply pathway, the biosensor having a space that encirclessurface of the filter in an area from one end of the filter close to thesample supply unit to the other end of the filter close to the electrodesystem.

[0041] Another aspect of the present invention is a biosensor, whichincludes: an insulating base plate, an electrode system that is providedon the base plate and has at least a working electrode and a counterelectrode, a cover member that is combined with the base plate, a samplesolution supply pathway that is formed between the cover member and thebase plate for leading a sample solution from a sample supply unit onthe cover member to the electrode system on the base plate, a reactionreagent system that includes at least an oxidation-reduction enzyme andan electron mediator and is provided on or in the vicinity of theelectrode system, and a filter disposed between the electrode system andthe sample supply unit in the sample solution supply pathway, thebiosensor having a space that encircles surface of the filter in an areafrom one end of the filter close to the sample supply unit to the otherend of the filter close to the electrode system.

[0042] In the above construction, the sample solution like blood addeddropwise to the sample supply unit is absorbed by the filter and flowsthrough the sample solution supply pathway toward the electrode systemand the reaction reagent layer, while the solid substance like thehemocytes is filtered out by the filter. Only the sample solution fromwhich the solid component like the hemocytes is filtered out accordinglyreaches the electrode system. In the sample solution supply pathwayclose to the sample supply unit, however, part of the sample solutionmay not be absorbed by the filter but may directly flow from a littlegap in the contact area of the filter with the sample solution supplypathway into the sample solution supply pathway. Even in such cases, thegap encircling the surface of the filter effectively prevents the samplesolution from further flowing toward the electrode system. Therefore,the sample solution including the solid substance like hemocytes isprevented from flowing through the space toward the electrode system. Itis preferable that the reaction reagent system is provided on or in thevicinity of the electrode system in the sample solution supply pathway.

[0043] A diversity of enzymes may be used for the oxidation-reductionenzyme included in the reaction reagent system. Such examples includeglucose oxidase, lactate oxidase, and cholesterol oxidase.

[0044] In the case of measurement of serum cholesterol, cholesteroloxidase and an enzyme having an ability of hydrolyzing cholesterol esterare used. Cholesterol esterase and lipoprotein lipase are examples ofthe enzyme having the ability of hydrolyzing cholesterol ester.Especially cholesterol esterase is preferable since it quickly changescholesterol ester to cholesterol in the presence of an appropriatesurface active agent.

[0045] When the reaction reagent system includes the enzyme having theability of hydrolyzing cholesterol ester, it is preferable that thereaction reagent system further includes a surface active agent forenhancing the ability of the enzyme. This desirably shortens the timerequired for the enzyme reaction.

[0046] Any of n-octyl-β-D-thioglucoside, polyethylene glycol monododecylether, sodium cholate, dodecyl-β-maltoside, sucrose monolaurate, sodiumdeoxycholate, sodium taurodeoxycholate, N,N-bis(3-D-gluconamidepropyl)cholamide, N,N-bis(3-D-gluconamide propyl)deoxycholamide,polyoxyethylene-p-t-octylphehyl ether (TritonX-100) may be used for thesurface active agent for enhancing the activity of cholesterol esterase.

[0047] When an electrochemically stable metal like platinum is appliedfor the electrode system of the biosensor, the observed value ofoxidation current does not include an error. Such metals are, however,expensive. In disposal-type sensors, the electrode system includes asilver electrode composed of, for example, silver paste, and a carbonelectrode obtained by covering the silver electrode with carbon paste.When the sample solution includes a surface active agent, the samplesolution soaks into carbon particles by the function of the surfaceactive agent. This may lower the activity of the carbon electrode. Thisalso causes the sample solution to be in contact with the silverelectrode. When a voltage is applied to the working electrode under suchconditions, an oxidation reaction may occur on the silver electrode togenerate electric current and give a positive error to the observedvalue of electric current.

[0048] One proposed method to suppress such phenomena covers the surfaceof the electrode system with a hydrophilic polymer. The hydrophilicpolymer makes the introduced sample solution a viscous layer, whichprevents the sample solution from coming into contact with theelectrodes.

[0049] Examples of the hydrophilic polymer include carboxymethylcellulose, polyvinyl pyrrolidone, polyvinyl alcohol, ethyl cellulose,hydroxypropyl cellulose, gelatin, polyacrylic acid and its salts, starchand its derivatives, polymers of maleic anhydride or its salts,polyacrylamide, methacrylate resin, and poly-2-hydroxyethylmethacrylate.

[0050] Other than the method using the hydrophilic polymer, thefollowing method may be applied to suppress the effects of the surfaceactive agent discussed above. A specific part of the electrode systemthat comes into contact with the sample solution is made of only carbonpaste. Silver paste for ensuring the electric conductivity ais used onlyfor the part covered with an insulating layer. The hydrophilic polymerlayer is not required in such printed electrodes. The hydrophilicpolymer, however, also functions to prevent protein in the samplesolution or the mixture of the sample solution and the reaction reagentfrom being adsorbed on the surface of the electrode and lowering theactivity of the electrode reaction. It is accordingly preferable to usethe hydrophilic polymer even in such printed electrodes.

[0051] When silver and carbon are used for the electrode system of thebiosensor, an electron mediator should be added to the reaction reagentlayer.

[0052] Any water-soluble compound that mediates transfer of electronsbetween the enzyme and the electrode, for example, potassiumferricyanide, p-benzoquinone, phenazine methosulfate, and ferrocenederivatives (oxidation type) may be applied for the electron mediator.

[0053] A two electrode system using only the working electrode and thecounter electrode and a three electrode system using the referenceelectrode in addition to the two electrodes is applicable to measure theoxidation current. The three electrode system is preferable to ensurethe higher accuracy of measurement.

[0054] The present invention is described in detail by referring to someembodiments. The drawings are only illustrative and the relativedimensions of the respective elements do not reflect the accurate sizes.

[0055]FIG. 1 is a decomposed perspective view illustrating a biosensorin one embodiment of the present invention, and FIG. 2 is a verticalsectional view of the biosensor.

[0056] Silver paste is printed on an insulating base plate 1 ofpolyethylene terephthalate by screen printing to form leads 2 and 3 andthe base of an electrode system. Electrically conductive carbon pastecontaining a resin binder is further printed on the base plate 1 to forman electrode system including a working electrode 4 and a counterelectrode 5, while insulating paste is printed to form an insulatinglayer 6. The working electrode 4 is connected to the lead 2, and thecounter electrode 5 to the lead 3. The insulating layer 6 makes theexposed areas of the working electrode 4 and the counter electrode 5constant, and partly covers the leads.

[0057] The insulating base plate 1 with the electrode system, a cover 8with an air vent 9, s spacer 7, and a filter 11 having the ability offiltering hemocytes are bonded according to the positional relationshipshown by the one-dot chain line, thereby to prepare a biosensor. Asample solution supply pathway running along the base plate 1 and thecover 8 is defined by a slit 10 of the spacer 7 between the base plate 1and the cover 8. The filter 11 is cut into the size fitting the samplesolution supply pathway and is disposed between the electrode system anda sample supply unit not to cover the electrode system. Numerals 13 aand 13 b represent parts at which the filter 11 is in contact with theinsulating base plate 1 and the cover 8, respectively.

[0058] There is a specific area from one end of the filter 11 close tothe sample supply unit 12 to the other end close to the electrodesystem, in which the surface of the filter 11 is not in contact with anyof the base plate 1, the spacer 7, and the cover 8 that define thesample solution supply pathway. In order to make this area, the baseplate 1 and the spacer 7 respectively have through holes 14 and 15 atcorresponding positions, and the cover 8 has two notches 16 connectingwith the slit 10. Lids 17 and 18 are attached to the outer faces of thebase plate 1 and the cover 8 to respectively cover the through holes 14and 15 formed in the base plate 1 and the cover 8. The through holes 14and 15 and the notches 16,16 form the space encircling the surface ofthe filter 11.

[0059] The through holes 14 and 15 can exert their functions even in theopen condition, although they are closed with the lids 17 and 18 in thisembodiment. The filter 11 is exposed to outside in the open condition.There is accordingly some possibility that evaporation of the samplesolution through the exposed part causes the liquid passing through thefilter and reaching the electrode system to flow back. The lids 17 and18 are accordingly provided to cover the through holes of the base plateand the cover. In the case where both the base plate and the cover aresufficiently thick, recesses that do not require the lids 17 and 18 maybe formed instead of the through holes.

[0060] When the sample solution is added dropwise onto the sample supplyunit 12 on the base plate to come into contact with an end of the filter11 close to the sample supply unit, the sample solution soaks into thefilter 11. The filter 11 removes the solid component like hemocytes, andthe plasma flows through the sample solution supply pathway and is ledinto the sensor. The plasma fills up the whole sample solution supplypathway from the vicinity of the electrode system to the portion of theair vent 9, while dissolving therein the reaction reagents carried at aspecific position covering over the electrode system or on the rear faceof the cover immediately above the specific position. When the wholesample solution supply pathway is filled with the liquid, the flow ofthe liquid in the filter 11 stops. At this moment, the hemocytes do notreach the end of the filter 11 close to the electrode system but areretained at the current position. Thus the filter 11 is designed toproduce such a difference in flow resistance between plasma andhemocytes that the hemocytes have not yet reached the end of the filterwhen the plasma has passed through the filter and filled up the wholesample solution supply pathway.

[0061] In this embodiment, the length between one end of the samplesolution supply pathway defined by the slit 10 on the sample supply unitto the outer circumference of the air vent 9 is 12.5 mm. The slit 10 hasthe width of 2.0 mm and the depth of 0.1 mm.

[0062] The dimensions of the through holes 14 and 15 expressed as(dimension in the direction perpendicular to the longitudinal directionof the base plate)×(dimension in the longitudinal direction of the baseplate) are 4.0×3.0 mm, and the dimensions of the notches 16 are also4.0×3.0 mm. Both the base plate and the cover have the thickness of 0.35mm, and the thickness of the spacer is 0.1 mm. Accordingly, the filter11 is surrounded by space having a thickness of 0.35 mm above and belowthe filter 11 and a thickness of 2.0 mm on both right and left sides ofthe filter 11, and a width of 3.0 mm in the flowing direction of thesample solution (hereinafter referred to as the width of the space). Thespace is located at a position 1 mm apart from the end of the samplesupply unit and 3.0 mm apart from the end of the electrode system. Theabove dimensions show just an example of the preferred embodiment, andare not restrictive in any sense.

[0063]FIG. 2 is a vertical sectional view illustrating the assembledbiosensor. A hydrophilic polymer layer 21 and an electron mediator layer22 covering over the hydrophilic polymer layer 21 are formed on theelectrode system on the base plate 1. The filter 11 is disposed in thesample solution supply pathway defined by the slit 10 of the spacer 7.The end of the filter 11 may be or may not be in contact with theelectrode system, but must not be in contact with and be apart from theworking electrode 4 in the electrode system. In the sample solutionsupply pathway, a layer 23 including enzymes and a surface active agentis formed in a specific area interposed between an end of the filter 11close to the electrode system and the air vent 9 on the rear face of thecover 8. The contact of this layer 23 with the end of the filter 11facilitates the flow of the sample solution into the layer 23, althoughthe contact is not essential.

[0064]FIG. 3 is a plan view illustrating the positional relationshipbetween the spacer and the filter in a biosensor in another embodimentof the present invention. The slit 10 for defining the sample solutionsupply pathway has a portion 10 a in which the filter is fitted, and aportion 10 b that has the electrode system and receives a flow-in samplepassing through the filter. The width of the portion 10 a is differentfrom the width of the portion 10 b. More specifically, in the embodimentof FIG. 3, the width of the portion 10 a with the filter fitted thereinis narrower than the width of the portion 10 b with the electrodesystem.

[0065]FIG. 4 is a vertical sectional view illustrating a biosensor instill another embodiment of the present invention. This biosensor has asimilar structure to that of FIG. 2 with a different arrangement of thereaction reagent layer. In the structure of this embodiment, only thehydrophilic polymer layer 21 is formed on the electrode system. A porouscarrier 24 impregnated with enzymes, a surface active agent, and anelectron mediator is provided on the cover 8 to be in contact with anend of the filter 11.

[0066]FIG. 5 is a vertical sectional view illustrating a biosensor inanother embodiment of the present invention, and FIG. 6 is a decomposedperspective view of the biosensor without its reaction reagent layer.

[0067] As in the structure of FIG. 1, leads 32 and 33, a workingelectrode 34 and a counter electrode 35 connecting with the respectiveleads, and an insulating layer 36 are formed on an insulating base plate31. A combination of multiple spacers 41, 43, 45, 47, and 49 with acover 52 is provided on the base plate 31. A filter 51 is interposedbetween the spacer 43 and the cover 52. A through hole 53 in the cover52 forms a sample supply unit. Through holes 42, 44, 46, 48, and 50formed in the spacers 41, 43, 45, 57, and 49 define a sample solutionsupply pathway running in the direction of gravity. Since the throughholes 46 and 50 in the spacers 45 and 49 have diameters greater than thediameter of the filter 51, spaces surrounding the filter 51 are formedaround the filter 51, as shown by numerals 55 and 56. The spacer 47 ispartly in contact with the outer circumference of the filter 51 tolocate the filter 51. The spacer 41 has an air vent 54 that connects theend of the sample solution supply pathway to the air. The samplesolution is introduced in the direction of gravity by means ofcapillarity into the sample solution supply pathway, which connects thethrough hole 53 disposed above the electrode system to function as thesample supply unit to the electrode system. The movement of the samplesolution stops when the plasma passing through the filter 51 reaches theelectrode system.

[0068] The thickness of the spacers 49 and 45 that specify the height ofthe spaces 55 and 56 surrounding the filter 51 is preferably not lessthan 100 μm. The reaction of the sample solution with the reagentsproceeds in the through hole 42 formed in the spacer 41. The preferablethickness of the spacer 41 ranges 100 to 200 μm. The orientation of thesample solution supply pathway in the direction of gravity enables thesample to pass through the filter by means of the gravity and quicklyreach the reaction reagent layer.

[0069] In this embodiment, a CMC layer 61 and an electron mediator layer62 are formed on the electrode system. A layer 63 including enzymes anda surface active agent is formed on the rear face of the spacer 43.

[0070]FIG. 7 is a vertical sectional view illustrating a biosensor inanother embodiment of the present invention. FIG. 8 is a decomposedperspective view illustrating the biosensor of FIG. 7 without a reactionreagent layer. This sensor is practically similar to the sensor shown inFIGS. 5 and 6, except that the spacer 43 is replaced by a samplesolution induction layer 57 mainly composed of non-woven fabric. In thebiosensor of this embodiment, a CMC layer 61 and a layer 64 includingenzymes, a surface active agent, and an electron mediator are providedon the electrode system.

[0071] The following describes examples of the present invention.

EXAMPLE 1

[0072] A cholesterol sensor was produced as an example of thebiosensors. The process first added a 0.5% by weight of aqueous solutionof sodium carboxymethyl cellulose (hereinafter referred to as CMC) asthe hydrophilic polymer dropwise onto the electrode system on theinsulating base plate 1 shown in FIG. 1 and dried the solution in a hotblast drier at 50° C. for 10 minutes, so as to form a CMC layer 21. Theprocess subsequently added 4 μl of an aqueous solution of potassiumferricyanide (corresponding to 70 mM potassium ferricyanide) as theelectron mediator dropwise on to the CMC layer and dried the solution inthe hot blast drier at 50° C. for 10 minutes, so as to form a potassiumferrocyanide layer 22.

[0073] The process then added 2 μl of a 2% by weight of ethanol solutionof Triton X-100 as the surface active agent dropwise into a recessdefined by the cover 8 and the slit 10 of the spacer 7, and dried thesolution at room temperature for 3 minutes, so as to form a layer of thesurface active agent. Triton X-100 was added to an aqueous solution inwhich cholesterol oxidase originating from Nocardia (EC1.1.3.6,hereinafter referred to as ChOD) and cholesterol esterase originatingfrom Pseudomonas (EC. 3.1.1.13, hereinafter referred to as ChE) weredissolved. The process added 1.5 μl of the solution mixture onto thesurface active agent layer, froze the layer with liquid nitrogen, anddried it in a Kjeldahl flask overnight in a freeze drier to give anenzyme/surface active agent layer 23 including 1 unit (U)/sensorcholesterol oxidase, 2.5 U/sensor cholesterol esterase, and 2% by weightof the surface active agent. The process then arranged a 2 mm×8 mmrectangular glass filter (GC50 manufactured by ADVANTEC LTD., thickness:0.19 mm) at the position shown in FIG. 1 not to be in contact with theworking electrode.

[0074] The through holes 14 and 15 and the notches 16, 16 were formed atthe specific position with the filter of the sample solution supplypathway as shown in FIG. 1. This defined the area in which the surfaceof the filter was not in contact with any of the insulating base plate,the spacer, and the cover defining the sample solution supply pathway.The dimensions of these elements are specified previously with referenceto FIG. 1.

[0075] As the sample solution, 20 μl of a whole blood sample was addeddropwise onto the sample supply unit 12 of the base plate 1. Visualobservations were made through the cover 8 composed of a transparentmaterial to confirm that the liquid passing through the filter reachedthe outer circumferential part of the air vent 9 in the sample solutionsupply pathway. At 3 minutes after the confirmation, a pulse voltage of+0.5 V was applied to the working electrode in the direction of theanode with the counter electrode as the reference. The value of electriccurrent was measured after 5 seconds. The result gave a responsedepending upon the concentration of cholesterol in serum.

[0076] The enzyme/surface active agent layer 23 was formed by the freezedrying method in this example, although it may be formed by the airdrying method. In the latter case, however, the solubility of thereaction reagent layer is significantly worsened. It accordingly takes along time until the reaction is completed after the filtered liquidreaches the outer circumference of the air vent 9 in the sample solutionsupply pathway.

EXAMPLE 2

[0077] In Example 1, the reaction reagent system was composed of theenzyme/surface active agent layer 23 formed by the freeze drying methodon the rear face of the cover in the sample solution supply pathway andthe CMC layer 21 and the potassium ferricyanide layer 22 formed by theair drying method at the specified position covering over the electrodesystem on the base plate. In this example, as shown in FIG. 4, thereaction reagent system was composed of a porous carrier 24, which wasin contact with an end of the filter 11 and had enzymes, a surfaceactive agent, and an electron mediator soaked therein and carriedthereon, and the CMC layer 21 formed by the air drying method at thespecified position covering over the electrode system on the base plate.

[0078] The arrangement of making part of the reagents included in thereaction reagent system carried on the porous carrier enhances thesolubility of the reaction reagents into the sample solution, as in thecase of the freeze drying method.

[0079] Like Example 1, the process first added a 0.5% by weight ofaqueous solution of CMC as the hydrophilic polymer dropwise onto theelectrode system and dried the solution in a hot blast drier at 50° C.for 10 minutes, so as to form the CMC layer 21.

[0080] The process bonded and fixed the porous carrier 24 made of feltwhich was cut into the size of 2×4.5 mm and mainly composed of glassfilters at a specified position shown in FIG. 4 on the cover in thesample solution supply pathway with a cellulose-based adhesive (CemedineC manufactured by Cemedine Co., Ltd), so as to be in contact with an endof the filter 11.

[0081] The process added 5 μl of the aqueous solution, in whichcholesterol oxidase, cholesterol esterase, potassium ferricyanide, andTriton X-100 were dissolved as in the case of Example 1, dropwise ontothe porous carrier 24, made the solution homogeneously soak into theporous carrier 24, and dried the solution in a hot blast drier at 50° C.for 15 minutes.

[0082] Like Example 1, the process located the filter and joined thecover member with the base plate 1 to complete a biosensor. The porouscarrier 24 had the thickness of approximately 0.1 to 0.2 mm. Thedistance between the base plate 1 and the cover 8 in the part of thesample solution supply pathway closer to the electrode system wasaccordingly set equal to 0.3 mm, which was significantly greater than0.1 mm in the structure of Example 1. In Example 2, GB1OOR was appliedfor the filter 11.

[0083] This biosensor showed a response corresponding to theconcentration of cholesterol at three minutes after the dropwiseaddition of the whole blood sample to the sample supply unit.

[0084] In the above examples, the base plate 1 and the cover 8 were madeof a transparent material, so that the flow-in state of the sample wasobservable with naked eyes.

[0085] In the above examples, the specific part of the slit 10 forforming the sample solution supply pathway with the filter fittedtherein has a width equal to the width of the part that has theelectrode system and receives a flow of the sample passing through thefilter. One of these parts may be narrower than the other. FIG. 5 showsthe positional relationship between the spacer and the filter and theirshapes in such an example.

[0086] The arrangement of the reagents constituting the reaction reagentlayer and their carrying method are not restricted to the specificationsof the above examples, as long as the reagents in the reaction reagentsystem are quickly dissolved in the sample solution to ensure smoothprogress of the enzyme reaction.

[0087] Industrial Applicability

[0088] As described above, the present invention effectively prevents asolid component like hemocytes included in a sample solution from cominginto contact with the electrode system and the reaction reagent systemand thereby provide a biosensor that ensures highly accurate measurementand has a little variation in response.

1. A biosensor, comprising: an insulating base plate, an electrodesystem that is provided on said base plate and has at least a workingelectrode and a counter electrode, a cover member that is combined withsaid base plate to define a sample solution supply pathway for leading asample solution from a sample supply unit to said electrode system, areaction reagent system including at least an oxidation-reduction enzymeand an electron mediator, and a filter disposed between said electrodesystem and said sample supply unit in said sample solution supplypathway, said biosensor having a space that encircles surface of saidfilter in an area from one end of said filter close to said samplesupply unit to the other end of said filter close to said electrodesystem.
 2. A biosensor in accordance with claim 1, wherein said covermember is disposed above said base plate, and said sample solutionsupply pathway starts from said sample supply unit provided on said baseplate and is formed along said cover member and said base plate.
 3. Abiosensor in accordance with claim 1, wherein said sample supply unit islocated on a side of said electrode system.
 4. A biosensor in accordancewith claim 3, wherein said space has a width of 0.5 mm to 5.0 mm.
 5. Abiosensor in accordance with claim 4, wherein said space has a width of1.0 mm to 3.0 mm.
 6. A biosensor in accordance with claim 1, whereinsaid sample solution supply pathway is disposed in a direction ofgravity from said sample supply unit provided on said cover member.
 7. Abiosensor in accordance with claim 1, wherein said sample supply unit islocated above said electrode system.
 8. A biosensor in accordance withclaim 6 or 7, wherein width of said space is not less than 100 μm andsmaller than thickness of said filter.
 9. A biosensor in accordance withclaim 5 or 8, wherein said filter is a porous body having spacesconnecting with one another in a three-dimensional manner, and saidporous body moves blood from said sample supply unit toward said samplesolution supply pathway by capillarity and functions to filter hemocytesbased on a difference between flow resistances of plasma and thehemocytes.
 10. A biosensor in accordance with claim 9, wherein theoxidation-reduction enzyme is cholesterol oxidase.
 11. A biosensor inaccordance with claim 10, wherein said reaction reagent system includesan enzyme having an ability of hydrolyzing cholesterol ester.
 12. Abiosensor in accordance with claim 11, wherein the enzyme having theability of hydrolyzing cholesterol ester is cholesterol esterase.
 13. Abiosensor in accordance with claim 11 or 12, wherein said reactionreagent system includes a surface active agent.
 14. A biosensor inaccordance with claim 13, wherein part or all of said cover member andsaid insulating base plate are transparent.